Synthetic virus-like particles with heterologous epitopes

ABSTRACT

The invention is a series of synthetic virus-like particles comprising a heterologous conformational epitope useful in the characterization of human papillomavirus infection, and useful to vaccinate individual for protection against HPV 6 and HPV 11 infections, and assays employing the synthetic virus-like particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/857,337, filed Jun.4, 2001, now U.S. Pat. No. 6,689,366. which is a 371 of PCT/US99/29577,international filing date of Dec. 14, 1999, which claims priority toU.S. Ser. No. 60/112,610, filed Dec. 17, 1998, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to recombinant papillomavirusvirus-like particles (VLPs) comprising heterologous neutralizingconformational epitopes. This invention also includes nucleic acidsencoding these VLPs and assays employing these synthetic VLPs.

BACKGROUND OF THE INVENTION

Human papillomavirus (HPV) types 6 and 11 are the causative agents formore than 90% of all genital condyloma and laryngeal papillomas. HPV isa DNA virus which is enclosed in a capsid which is made up principallyof L1 protein. The L1 proteins of HPV types 6 and 11 are very similar atboth the amino acid and nucleotide level. Consequently, it has beendifficult to develop assays which reliably distinguish between these twotypes of infection.

HPV 11 L1 residues Gly¹³¹-Tyr¹³² were previously identified asresponsible for the type-specific binding of several HPV 11 neutralizingmonoclonal antibodies (Ludmerer et. al. 1996. “Two Amino Acid ResiduesConfer Type Specificity to a Neutralizing, Conformationally DependentEpitope on Human Papillomavirus Type 11”. J. Virol. 70:4791–4794).Within this same work, it was further demonstrated that a substitutionat Ser³⁴⁶ of the HPV 11 L1 sequence dramatically reduced binding ofneutralizing monoclonal antibody H11.H3, and that the effect wasspecific for this antibody. Additional studies demonstrated that severalHPV 11 neutralizing antibodies bound to a stretch of the HPV 11 L1sequence between residues 120–140, whereas H11.H3 bound to a completelydistinct site (Ludmerer et al. 1997. “A Neutralizing Epitope of HumanPapillomavirus Type 11 is Principally Described by a Continuous Set ofResidues Which Overlap a Distinct Linear, Surface-Exposed Epitope”. J.Virol. 71:3834–3839).

However, these studies did not define which amino acid residues confertype specificity of binding for antibody H11.H3 completely. Furthermore,there may be other regions of HPV 11 VLPs, not described in thesestudies, which can elicit important HPV 11-specific, conformationallydependent responses. In addition, VLP-dependent antibodies specific forHPV 6 have also been generated (Christensen et al. 1996 “MonoclonalAntibodies to HPV-6 L1 Virus-Like Particles Identify Conformational andLinear Neutralizing Epitopes on HPV-11 in Addition to Type-SpecificEpitopes on HPV-6”. Virology 224(2):477–486). These antibodies could beuseful in evaluation of infectivity by HPV 6. It would be desirable todetermine the exact amino acids involved in the specificity of HPV type6- and additional type 11-specific conformational epitope formations sothat improved assays and vaccines may be developed.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a recombinant papillomavirus L1 protein ofa first subtype which comprises a conformational epitope of apapillomavirus L1 protein of a second subtype. Preferably, the L1protein is part of a virus-like particle (VLP). In some embodiments, thepapillomavirus is a human papillomavirus (HPV). In a specific embodimentof this invention, a human papillomavirus L1 protein comprises aheterologous conformational epitope from HPV 6. In another specificembodiment of this invention, a human papillomavirus L1 proteincomprises a heterologous conformation epitope from HPV 11.

Another aspect of this invention are nucleic acids encoding theseheterologous L1 proteins, particularly DNA.

Another aspect of this invention are assays employing the syntheticvirus-like particles.

Another aspect of this invention are vaccines comprising nucleic acidsand/or proteins encoded by the nucleic acids, wherein the proteinscomprise a heterologous conformational epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences for the L1 protein of HPV 6 andHPV 11 mutants utilized in these studies.

FIG. 2 are graphs which show that amino acid substitutions at criticalpositions within the HPV 6 L1 sequence eliminate binding of monoclonalantibodies H6.B10.5, H6.M48, and H6.N8. In the first graph, the leftmost bar is H6.B10.5; the second bar is H6,.M48; the third bar is H6.C6and the right most bar is H6.J54. In the second graph the left bar isH6.N8 and the right bar is H6.C6.

FIG. 3 shows substitutions into the HPV 11 L1 sequence which conferbinding of HPV 6 specific monoclonal antibodies H6.B10.5, H6.M48, andH6.N8. The left most bar is H6.B10.5; the next bar is H6.M48; the nextbar is H6.N8 and the right most bar is H6.C6.

FIG. 4 shows that three amino acid substitutions into the HPV 6 L1sequence between residues 345 and 348 confer binding of HPV 11monoclonal antibodies H11.G3 and H11.H3. It also shows that sevensubstitutions between residues 262 and 289 also confer binding of HPV11monoclonal H11.G3. The left most bar is H11.H3; the second bar isH11.B2; the third bar is H11.G3; and the right most bar is H11.C6.

FIG. 5 shows that three substitutions into the HPV 6 sequence betweenresidues 49 and 54, or four substitutions into the HPV 6 sequencebetween residues 169 and 178 confer binding of HPV 11 monoclonalantibody H11.A3.2. The left most bar is H11.A3.2; the middle bar isH11.B2 and the right bar is H11.C6.

As used throughout this specification and claims, amino acid residues(wild-type) are referred to by a two-part designation which is (i) theone-letter standard amino acid abbreviation of the wild-type residue,followed by (ii) the position of the amino acid in the L1 protein.Residues specified in this format would also be for a particular HPVtype. For example, “HPV 6 K53” means the lysine residue at position 53of HPV 6 L1.

As used throughout this specification and claims, mutated amino acidsare referred to by a three-part designation which is (i) the one-letterstandard amino acid abbreviation of the wild-type amino acid (ii) theposition of the amino acid in the L1 protein of a particular HPV type,and (iii) the one letter abbreviation for the amino acid which is nowpresent. For example, HPV 6 “T345S” means that the threonine residue,normally present at position 345 of HPV 6 L1, has been changed toserine.

Monoclonal antibodies referred to throughout this specification arelisted below (all were obtained from Dr. Neil Christensen ofPennsylvania State University, Hershey, Pa.).

H6.B10.5—this antibody is specific to HPV 6 L1 protein in VLPs.

H6.M48—this antibody is similar to H6.B10.5 in that it binds to HPV 6 L1protein in VLPs.

H6.N8—this antibody is similar to H6.B10.5 in that it binds to HPV 6 L1protein in VLPs.

H11.G3—this antibody is specific for HPV 11 VLPs and its binding isconformationally dependent.

H11.H3—this antibody is specific for HPV 11 VLPs, and is neutralizing.It is known that HPV 11 L1 residue S346 is critically important for thebinding of H11.H3, and that a substitution at this position does notaffect binding of HPV 11-specific VLP-dependent antibodies demonstratedto bind elsewhere.

H6.J54—this antibody binds VLPs of both HPV 6 and HPV 11, but not otherHPV types.

H6.C6—this antibody binds both HPV 6 and 11 VLPs; it also binds bothnative and denatured material. It can be used to determine total L1production.

H11.A3—this antibody specifically binds HPV 11 VLPs, but at a differentregion than either H11.B2 or H11.H3.

H11.B2—similar to H11.A3 in that it specifically binds HPV 11 VLPs, butat a different region than either H11.H3 or H11.A3.

In order to develop an assay which would distinguish between HPV 6 andHPV 11 responses, the amino acids residues that confer antigenictype-specificity on HPV subtypes had to be determined. Therefore wefocused on regions of HPV 6 which have multiple divergences from HPV 11within a short stretch. To streamline the process of analyses, mutantsof HPV 6 which were multiply mutated at these regions were synthesizedand the VLPs produced from them were analyzed for effects on antibodybinding.

In order to construct the VLPs of this invention, the amino acidresidues which make up the conformational epitopes where type specific,VLP-dependent monoclonal antibodies bind had to be determined. This wasaccomplished by mapping the binding sites of monoclonal antibodies whichbind specifically to either HPV 6 or HPV 11 VLPs. L1 of either HPV 6 orHPV 11 was modified by introducing amino acid substitutions at variouspositions, then determining if the mutant protein would bind either HPV6- or HPV 11-specific monoclonal antibodies. Mapping was confirmed bydemonstrating transfer of binding of a monoclonal to one of these typesto the other type which had been minimally modified. Modified VLPs usedfor these transfers were demonstrated to retain binding of othertype-specific antibodies.

In accordance with this invention it has been found that HPV 6 L1 withthree HPV 11 L1-like substitutions, at T345, T346 and S348 produce VLPsthat bind HPV 11-specific monoclonal antibodies H11.G3 and H11.H3. Thesetwo antibodies can be distinguished in that H11.G3, but not H11.H3, canalso bind HPV 6 VLPs which contain seven substitutions between residues262 and 290. In specific embodiments of this invention, thesubstitutions are T345S, T346K, and S348A.

Furthermore, we show that HPV 6 L1 with either three HPV 11 L1-likesubstitutions between amino acids 49 and 54 (at F49, R53, and A54), orfour HPV 11 L1-like substitutions between residues 169 and 178 (at K169,T172, P175, and A178), can bind HPV 11-specific, VLP dependentmonoclonal antibody H11.A3.2. In specific embodiments of these class ofmutants, the substitutions are (i) the combination of F49Y, R53K, andA54V; and (ii) K169T, T172S, P175S, and A178N.

Thus one aspect of this invention is a recombinant HPV 6 L1 proteinwhich also presents a major neutralizing, conformational epitope of HPV11. In a preferred embodiment, the conformational epitope comprisesT345S, T346K and S348A. These whole regions may be transferred to otherHPV types through alignment, generating more refined tools forserological analysis. Thus this invention comprises any papillomavirustype which comprises a heterologous neutralizing conformational epitopeof HPV 11 mapped in these studies.

This invention also includes HPV 6 L1 proteins with HPV 11-likesubstitutions between residues 49–54, 169–178, and 261–290, specificallyat (i) F49, R53, and A54; (ii) K169, T273, P175 and A178; and (iii)E262, T270, S276, G277, T280, G283, and N289. In specific embodiments ofthis class of mutants, the substitutions are: (i) F49Y, R53K, and A54V;(ii) K169T, T273S, P175S, and A178N; and (iii), E262T, T270D, S276G,G277N, T280S, G283A, and N289H. These portions of the protein comprisepart of the epitope for HPV 11-specific VLP dependent monoclonalantibodies H11.A3.2, and H11.G3.

A further aspect of this invention is nucleic acids encoding the L1proteins comprising the heterologous conformational epitopes discussedabove, including HPV 11 conformational epitope T345S, T346K and S348A.As L1 protein and nucleic acid sequences are generally well known, it iswithin the skill of the ordinary artisian to insert the mutationsdescribed herein using conventional genetic engineering/proteinengineering techniques. In a preferred embodiment the nucleic acid is aDNA, and codons may be optimized for increased viral expression for agiven host cell.

Other aspects of this invention include vectors such as plasmids whichcontain the nucleic acids encoding L1 proteins comprising a heterologousHPV 11 conformational epitope. Also included in this invention are hostcells, particularly yeast, bacterial, insect, and mammalian cellscontaining a nucleic acid encoding an L1 protein comprising an HPV 11conformational epitope, whether or not present in a vector.

In another aspect of this invention, HPV 6 epitopes were transferred toHPV 11. In this embodiment, HPV 11 L1 modified with either one (at K53)or two ( at Y49 and K53) substitutions show approximately a 10-foldincrease in binding of HPV 6-specific monoclonal antibody H6.N8. In thecase of H6.N8, the level of binding was comparable to that observed withprototype HPV 6 VLPs. This demonstrates that part of the epitope,including its type 6 specificity, is defined by these and neighboringresidues. Preferred substitutions are K53R, and the combinantion of Y49Fand K53R.

Another HPV 11 heterologous L1 protein comprises three changes: at Y49,K53, and V54. HPV 11 L1 modified with these three changes showapproximately four-fold binding above background to HPV 6-specificmonoclonal antibodies H6.B10.5 and H6.M48, in addition to bindingmonoclonal antibody H6.N8 as discussed above. In specific embodiments ofthis class of mutants, the substitutions are Y49F, K53R, and V54A.

Another HPV 11 L1 mutant encompasses seven changes in two regions (49–54and 170–179). These changes are at positions Y49, K53, V54, T170, S173,S176, and N179. HPV 11 L1 modified with these seven substitutions showbinding to antibodies H6.B10.5, H6.M48, and H6.N8 comparable to thatobserved with prototype HPV 6 VLPs. These HPV 6 epitopes can be moved toany desired PV type. In preferred embodiments the substitutions are HPV6 residues F49, R53, A54, K169, T172, P175, and A178, placed intoequivalent positions of the selected PV L1 type by alignment.

Another aspect of this invention is nucleic acids encoding the L1proteins comprising a heterologous conformational epitope, including (i)HPV 6 conformational epitope K53R, (ii) the combination of Y49F andK53R, (iii) the combination of Y49F, K53R, and V54A, (iv) thecombination of Y49F, K53R, V54A, T170K, S173T, S276P and N179A; and (v)combinations of (i), (ii), (iii) and/or (iv). As L1 protein and nucleicacid sequences are generally well known, it is within the skill of theordinary artisian to insert the mutations described herein usingconventional genetic engineering/protein engineering techniques. In apreferred embodiment the nucleic acid is a DNA, and codons may beoptimized for increased viral expression for a given host cell. Otheraspects of this invention include vectors such as plasmids which containthe nucleic acids encoding L1 proteins comprising a heterologous HPV 6conformational epitope. Also included in this invention are host cells,particularly yeast, bacterial, insect, and mammalian cells containing anucleic acid encoding an L1 protein comprising an HPV 6 conformationalepitope, whether or not present in a vector.

In another aspect of this invention is the transfer of conformationalepitopes to a more distantly-related PV type. Almost every species ofanimal studied to date has a PV, including cottontail rabbit, bovine,canine, and the like. This discussion and the examples focus on the useof cottontail rabbit papillomavirus (CRPV) comprising heterologousconformational epitopes to produce serological reagents of higherspecificity to monitor HPV 6 and HPV 11 responses and infectivity.However, it is intended that any PV can substitute for CRPV for theseuses, and the present invention is specifically directed to this broadusage. Thus, this invention specifically includes a recombinant CRPV L1protein comprising at least one heterologous conformational epitope. Inspecific embodiments, the heterologous conformational epitope isselected from the groups consisting of human HPV 6 and HPV 11 epitopes,and placed into CRPV L1 by amino acid alignment. This invention alsoincludes nucleic acids encoding the recombinant protein, vectorscomprising the nucleic acids, and host cells which comprise the vectors.

A further aspect of the invention is the generation of VLPs which canelicit both HPV 6 and HPV 11 responses. HPV 6 VLPs which contain HPV 11substitutions T345S, T346K, and S348A will present a major HPV 11neutralizing epitope alongside all HPV 6 responses. HPV 11 VLPs modifiedto contain HPV 6 substitutions, such as the combination of Y49F andK53R; the combination of Y49F, K53R, and V54A; or the combinantion ofY49F, K53R, V54A, T170K, S173T, S176P and N179A will present the oneknown HPV 6 specific conformational epitope alongside most HPV 11epitopes, including the major neutralizing epitopes. The latter issignificant in that all known neutralizing epitopes are conformationallydependent, and conformational dependence is believed to be a necessaryproperty of such epitopes.

Because of the high identity between wild-type HPV 6 and HPV 11 L1sequences, present serological assays cannot distinguish responsesbetween these two types very well. The modified VLPs of this inventionwill identify HPV 6 and HPV 11 immune responses upon infectivity orimmunization. VLPs which elicit neutralizing responses to both types cansimplify vaccine manufacturing, and lower costs for the consumer.

Another aspect of this invention is the use of these derivatized HPV 6and HPV 11 VLPs as reagents in serological assays. Because most epitopesare shared between HPV 6 and HPV 11 VLPs, polyclonal sera to onecompetes with the binding of a type-specific monoclonal antibody to theother due to steric hindrance from the binding of antibodies toneighboring sites. In accordance with this invention, HPV 6 and HPV 11epitopes can be moved to a more distant VLP type, such as CRPV, wherethere are no cross-reactive epitopes between CRPV and either HPV 6 orHPV 11. Therefore, presentation of HPV 6- and HPV 11-specific epitopeson a CRPV VLP eliminates the problem of steric competition fromneighboring epitopes. Only the presence of antibody in a polyclonalresponse to the specifically transferred epitope should compete withmonoclonal antibody binding.

One assay of this invention distinguishes between the presence of HPV 6and HPV 11 antibodies in a sample suspected of containing either or bothtypes of antibodies comprising the steps of:

a) contacting the sample with recombinant PV protein comprising either aheterologous HPV conformational epitope or a heterologous HPV 11conformational epitope; and

b) detecting binding between the antibodies present in the sample andthe recombinant PV protein;

wherein binding to a heterologous HPV 11 conformational epitopeindicates the presence of HPV 11 antibodies in the sample, and bindingto a HPV 6 conformational epitope indicates the presence of HPV 6antibodies in the sample.

Yet another aspect of this invention comprises an assay fordiscrimination between HPV 6 and HPV 11 in a subject suspected of beinginfected with either HPV 6 or HPV 11, or vaccinated for protectionagainst HPV 6 and/or HPV 11 infection, comprising the steps of:

a) obtaining a blood sample from the subject, wherein said blood samplecomprises either HPV 6 or HPV 11 antibodies;

b) contacting the sample with a recombinant VLP comprising either aheterologous HPV 6 conformational epitope or a heterologous HPV 11conformational epitope; and

c) detecting binding between the antibodies present in the sample andthe heterologous VLP;

wherein binding a VLP comprising a heterologous HPV 11 conformationalepitope indicates an HPV 11 infection, and binding to a VLP comprising aheterologous HPV 6 conformational epitope indicates an HPV 6 infection.In preferred embodiments, the VLP is from a distantly related PV, suchas a CRPV VLP.

Another aspect of this invention are vaccines which comprise either L1protein comprising a heterologous conformational epitope, or the nucleicacid which encode these proteins. In specific embodiments, the proteincomprises both an HPV 6 and an HPV 11 epitope; either or both may beheterologous. This vaccine will confer protection against both types ofHPV infection, as neutralizing antibodies to both viral epitopes areproduced. The protein-based vaccine may be formulated according toconventional vaccine formulation techniques, and include such wellknown, traditional components as adjuvants, and pharmaceuticallyacceptable carriers. This vaccine may be administered intranasally,intravenously, intramuscularly, or subcutaneously, either with orwithout a booster dose. Likewise, nucleic-acid based vaccines, orspecifically, DNA vaccines may be similarly formulated and administered.

The following examples are provided to further define the inventionwithout, however, limiting the invention to the particulars of theseexamples.

EXAMPLE 1 Generation of Test Expression Constructs

The HPV 6 and HPV 11 L1 structural genes were cloned from clinicalisolates using PCR with primers designed from the published L1 sequence.The L1 genes subsequently were subcloned both into BlueScript(Pharmacia) for mutagenesis, and pVL1393 (Stratagene) for expression inSf9 cells.

Mutations were introduced into the L1 gene using Amersham Sculptor invitro mutagenesis kit according to the manufacturer's recommendations.The appearance of the desired mutation was confirmed by sequencing, andthe mutated gene subcloned into pVL1393 for expression in Sf9 cells.

EXAMPLE 2 Transient Expression of L1 VLPs in SF9 Cells

SF9 cells were transfected using BaculoGold Transfection kit(Pharmingen). Transfections were done essentially according to themanufacturer's instructions with the following modifications. 8×10⁸ Sf9cells were transfected in a 100 mM dish, with 4 μg of BaculoGold DNA and6 μg of test DNA. Cells were harvested after 6 days and assayed for VLPproduction.

EXAMPLE 3 Preparation of SF9 Extracts and ELISA Assays

Cells were harvested six days after transfection, by scraping followedby low speed centrifugation. Cells were resuspended in 300 ml ofbreaking buffer (1 M NaCl, 0.2 M Tris pH 7.6) and homogenized for 30minutes on ice using a Polytron PT 1200 B with a PT-DA 1205/2-A probe(Brinkman) in a Falcon 1259 tube. Samples were spun at 2500 rpm for 3minutes to pellet debris. Tubes were washed with an additional 150 ml ofbreaking buffer, supernatants collected in a 1.5 ml microfuge tube, andrespun for 5 minutes in an Eppendorf microfuge (Brinkman). Supernatantswere collected and stored at 4 C. until use. ELISA assays typically wereperformed the same day, although samples may be frozen on dry ice,stored at −80 C., thawed and assayed at convenience.

5 ml of extract was diluted into 50 ml of 1% BSA in PBS (phosphatebuffered saline; 20 mM NaPO₄, pH 7.0, 150 mM NaCl) and plated onto apolystyrene plate. The plate was incubated overnight at 4 C. Extractswere removed and the plate blocked with 5% powdered milk in PBS. Allsubsequent wash steps were performed with 1% BSA in PBS. The plate wasincubated at room temperature with primary antibody for 1 hour. Primaryantibodies (monoclonal antibodies generated against HPV 11 virions, HPV11 VLPs, or HPV 6 VLPs) were obtained as ascites stock from Dr. NeilChristensen (Pennsylvania State University). They were diluted 10⁵-foldin 1% BSA PBS before use. After washing, plates were incubated for 1hour with secondary antibody. The secondary antibody, peroxidase labeledGoat anti-Mouse IgG (γ), was purchased from Kirkegaard & PerryLaboratories, Inc. and used at 10³ dilution in 1% BSA in PBS. After afinal washing, a horse radish peroxidase assay was performed andabsorbance read at 450 nm.

EXAMPLE 4 Two Near Adjacent Substitutions into the HPV 6 L1 SequenceEliminates Binding of HPV 6-specific, VLP-dependent MonoclonalAntibodies

We predicted that an HPV 6-specific monoclonal antibody (one which doesnot bind to closely related HPV 11 VLPs) would bind a region where thereare several adjacent or near adjacent residues between types 6 and 11 L1genes. Excluding the C-terminus (it has been shown that the C-terminusis non-essential for VLP formation), there are five such regions. Usingstandard procedures, we generated test clones which had multiple 11-likesubstitutions in each of these five regions. Only clone 1393:6:49–53,which harbors substitutions at L1 residues 49 and 53 (F49Y, R53K)produced VLPs which had an effect on H6.B10.5, H6.M48, and H6.N8binding. Binding of HPV 11-cross-reactive antibody H6.J54, alsoVLP-dependent, was not disturbed, demonstrating the presence of VLPs.

EXAMPLE 5 Transfer of Binding of H6.B10.5, H6.M48 and H6.N8 to ModifiedHPV 11 VLPs

Based upon the studies in Example 4, we generated mutants of HPV 11 L1with HPV 6-like substitutions at positions within the first 60 residueswhere the two L1 sequences differ. We also generated HPV 11 mutants withsingle substitutions at either Y49F or K53R. A third HPV 11 cloneharbored three HPV 6-like substitutions between residues 49 to 54 (Y49F,K53R, and V54A), and a fourth clone harbored three HPV 6-likesubstitutions between residues 49 to 54 and 4 substitutions betweenresidues 170 to 180 (Y49F, K53R, V54A and T170K, S173T, S176P, N179A).

Clones 1393:11:K53R and 1393:11:Y49F,K53R both generated VLPs whichproduced approximately ten-fold binding above background of HPV6-specific monoclonal antibody H6.N8. An additional clone,1393:11:Y49F,K53R,V54A, generated VLPs which showed approximatelyfour-fold binding above background of monoclonal antibodies H6.B10.5 andH6.M48.

Antibody H6.C6 is cross-reactive between types 6 and 11 L1 , and bindsboth native and denatured material. Thus it is a measure of total L1production. Normalized to L1 production, the level of H6.N8 binding wascomparable to that observed with prototype HPV 6 VLPs. VLPs producedfrom clone 1393:11:49–54, 170–180, which harbored seven HPV 6-likesubstitutions over two distinct areas of L1 (Y49F, K53R, V54V; andT170K, S173T, S176P, N179A) showed a level of binding to antibodiesH6.B10.5, H6.M48, and H6.N8 which was comparable to that observed withprototype HPV 6 VLPs.

Antibodies H11.B2 and H11.H3, both type 11-specific and VLP-dependent,are known to bind other regions of the L1 sequence. Hence thesesubstitutions at the N-terminus should not impact their binding. Theybound these N-terminally mutated constructs, thus demonstrating thatthese N-terminal substitutions had no effect on VLP assembly, or on thepresentation of critical HPV 11 neutralizing epitopes.

This result is especially significant in light of the fact that thebinding site of antibody H11.B2 previously was mapped to a stretch ofresidues between Y123 and V142, a region which lies in between the twomultiply mutated regions discussed in the present example. Thisdemonstrates that the structural perturbations generated by themutations discussed in this work are quite localized.

EXAMPLE 6 Three Substitutions into HPV 6 L1 Sequence Confer H11.G3 andH11.H3 Binding

HPV 6 L1 was modified with HPV 11-like substitutions to generate1393:6:T345S,T346K, and A348S. This clone was expressed transiently inSf9 cells, and VLPs were produced and tested for binding for bothantibodies H11.G3 and H11.H3. We observed binding 10-fold abovebackground levels, commensurate with binding to prototype HPV 11 VLPs.Binding of HPV 6-specific antibodies H6.B10.5 and H6.M48 was notperturbed, demonstrating that the VLPs retained HPV 6-like character.Furthermore, binding of HPV 11-specific antibodies H11.A3 and H11.B2,antibodies known to bind elsewhere, was not observed, thus demonstratingthat the transfer was specific to H11.G3 and H11.H3.

EXAMPLE 7 Seven Substitutions into HPV 6 L1 Sequence Confer H11.G3Binding

HPV 6 L1 was modified with seven HPV 11-like substitutions betweenresidues 262 and 289 (E262T, T270D, S276G, G277N, T280S, G283A, N289H)to generate clone 1393:6:262–289. This clone was expressed transientlyin Sf9 cells, and VLPs were produced and tested for binding. We observedbinding 10-fold above background levels of antibody H11.G3. Binding ofHPV 6 specific antibodies H6.B10.5 and H6.M48 was not perturbed,demonstrating that the VLPs retained HPV 6-like character. Furthermore,binding of HPV 11-specific antibodies H11.A3 and H11.B2, antibodiesknown to bind elsewhere, was not observed, thus demonstrating that thetransfer was specific to H11.G3.

EXAMPLE 8 Three Substitutions into HPV 6 L1 Sequence Between Residues 49and 54, or Four Substitutions Between Residues 169 and 178, conferH11.A3.2 Binding

HPV 6 L1 was modified with three HPV 11-like substitutions betweenresidues 49 and 54 (F49Y, R53K, and A54V and four HPV 11-likesubstitutions between residues 169 and 178 (K169T, T172S, P175S, andA178N), or four HPV 11-like substitutions between residues 169 and 178(K169T, T172S, P175S, and A178N) to generate clones 1393:6:49–54,169–178 and 1393:6:169–178 respectively. These clones were expressedtransiently in Sf9 cells, and VLPs were produced and tested for binding.We observed binding three-fold above background for antibody H11.A3.2with either clone. Binding of HPV 6-specific antibodies H6.B10.5 andH6.M48 was not perturbed by clone 1393:6:169–178, demonstrating thatthese VLPs retained HPV 6-like character. Work described in thisdocument demonstrates that HPV 6- specific antibodies target region49–54, therefore it is expected that VLPs produced from clone1393:6:49–54 will not bind these antibodies. The binding of HPV11-specific antibody H11.B2, known to bind elsewhere, was not observed,thus demonstrating that the transfer was specific to H11.A3.2.

EXAMPLE 9 Monitoring Serological Responses to HPV 11 Infection orImmunization

HPV 6-modified VLPs are used to determine the presence of an immuneresponse to HPV 11 following viral infection or immunization with HPV 11VLPs. HPV 6-modified VLPs which present the HPV 11 neutralizing epitopeto H11.G3 and/or H11.H3 are coated onto the well of a microtitre platein native form. Following blocking, HPV 11 monoclonal antibody H11.G3and/or H11.H3 is incubated in ELISA format with increasing amounts ofHPV 11 polyclonal sera, HPV 6 polyclonal sera, and test polyclonal sera.Binding of the HPV 11 monoclonal antibody is visualized using a rabbitanti-mouse IgG secondary antibody. Alternatively, it is labeled withI¹²⁵, or coupled directly to horseradish peroxidase or alkalinephosphatase, or another standard ELISA visualization protocol. Anincreasing amount of polyclonal HPV 11 sera competes with binding untilthe signal eventually is reduced to background level. Polyclonal HPV 6sera does not compete, or the competition is significantly reduced fromthat observed with HPV 11 polyclonal sera. Competition with the testsera at levels comparable to HPV 11 polyclonal sera demonstrates animmune response to HPV 11. Lack of, or a significant reduction ofcompetition demonstrates lack of or a weak immune response to HPV 11.

EXAMPLE 10 Monitoring Serological Responses to HPV 6 Infection orImmunization

HPV 11-modified VLPs are used to determine the presence of an immuneresponse to HPV 6 following viral infection or immunization with HPV 6VLPs. HPV 11 modified VLPs which present the HPV 6 epitope to H6.N8and/or H6.M48 are coated onto the well of a microtitre plate in nativeform. Following blocking, HPV 6 monoclonal antibody H6.N8 and/or H6.M48is incubated in ELISA format with increasing amounts of HPV 11polyclonal sera, HPV 6 polyclonal sera, and test polyclonal sera.Binding of the HPV 6 monoclonal antibodies H6.N8 and/or H6.M48 isvisualized using a rabbit anti-mouse IgG secondary antibody.Alternatively, they are labeled with I¹²⁵, or coupled directly tohorseradish peroxidase or alkaline phosphatase, or another standardELISA visualization protocol. Increasing amounts of polyclonal HPV 6sera should compete with binding until the signal eventually is reducedto background level. Polyclonal HPV 11 sera does not compete.Competition with the test sera at levels comparable to HPV 6 polyclonalsera demonstrates an immune response to HPV 6. Lack of or significantreduction of competition demonstrates lack of or a weak immune responseto HPV 11.

EXAMPLE 11 Generation of Chimeric VLPs Which Stimulate Both Type 6 andType 11 Specific Responses

HPV 6 VLPs modified to contain substitutions, S131G and Y132, T345S,T346S, and S348A, produce VLPs which present i) the HPV 6-specific andVLP dependent epitope and ii) all known HPV 11 specific and neutralizingepitopes. Alternatively, HPV 11 VLPs modified to contain substitutionK53R, or Y49F and K53R, or Y49F, K53R, V54A, T170K, S173T, S176P, N179Aproduce VLPs which present i) the HPV 6-specific and VLP dependentepitope and ii) the major HPV 11 specific and neutralizing epitopes.These latter chimeric VLPs present the one type 6 specific epitopeknown, the two neutralizing type 11 epitopes known, and the 6/11 commonepitopes. Chimeric 6/11 VLPs are able to replace double immunizationwith type 6 and type 11 VLPs to stimulate immune responses, with reducedproductivity costs.

1. A method to distinguish between the presence of human papillomavirus6 (HPV 6) and human papillomavirus 11 (HPV 11) antibodies in a samplesuspected of containing either or both types of said antibodiescomprising the steps of: a) contacting the sample with a modifiedrecombinant human papillomavirus 6 (HPV6) L1 protein which comprises anHPV 11 conformational epitope; wherein the modified HPV 6 proteincomprises a mutation at locations selected from the group consisting of:(i) K169, T172, P175, and A178; (ii) T345, T346, and S348; (iii) F49,R53, and A54; and (iv) E262, T270, S276, G277, T280, G283 and N289; andb) detecting binding between the antibodies present in the sample andthe modified HPV6 protein; wherein binding to a heterologous HPV 11conformational epitope indicates the presence of HPV 11 antibodies inthe sample, and binding to a HPV 6 conformational epitope indicates thepresence of HPV 6 antibodies in the sample.
 2. A method according toclaim 1 wherein the modified HPV6 protein is a virus-like protein (VLP).3. A method according to claim 1 wherein the sample is a blood samplefrom an individual suspected of being infected with either HPV 6 or HPV11.